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Comparative Study
. 2010 Aug 26:10:459.
doi: 10.1186/1471-2407-10-459.

The PI3K/Akt pathway upregulates Id1 and integrin α4 to enhance recruitment of human ovarian cancer endothelial progenitor cells

Yajuan Su  1 , Lei Zheng, Qian Wang, Jie Bao, Zhen Cai, Ailan Liu
Affiliations
Comparative Study

The PI3K/Akt pathway upregulates Id1 and integrin α4 to enhance recruitment of human ovarian cancer endothelial progenitor cells

Yajuan Su et al. BMC Cancer. .

Abstract

Background: Endothelial progenitor cells (EPCs) contribute to tumor angiogenesis and growth. We aimed to determine whether inhibitors of differentiation 1 (Id1) were expressed in circulating EPCs of patients with ovarian cancer, whether Id1 could mediate EPCs mobilization and recruitment, and, if so, what underlying signaling pathway it used.

Methods: Circulating EPCs cultures were from 25 patients with ovarian cancer and 20 healthy control subjects. Id1 and integrin α4 expression were analyzed by real-time reverse transcription-polymerase chain reaction and western blot. EPCs proliferation, migration, and adhesion were detected by MTT, transwell chamber, and EPCs-matrigel adhesion assays. Double-stranded DNA containing the interference sequences were synthesized according to the structure of a pGCSIL-GFP viral vector and then inserted into a linearized vector. Positive clones were identified as lentiviral vectors that expressed human Id1 short hairpin RNA (shRNA).

Results: Id1 and integrin α4 expression were increased in EPCs freshly isolated from ovarian cancer patients compared to those obtained from healthy subjects. siRNA-mediated Id1 downregulation substantially reduced EPCs function and integrin α4 expression. Importantly, Inhibition of PI3K/Akt inhibited Id1 and integrin α4 expression, resulting in the decreasing biological function of EPCs.

Conclusions: Id1 induced EPCs mobilization and recruitment is mediated chiefly by the PI3K/Akt signaling pathway and is associated with activation of integrin α4.

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Figures

Figure 1
Figure 1
Phenotypic characterization of EPCs from human peripheral blood. After a week in culture, EPCs were stained with DIL-labeled ac-LDL, FITC-conjugated Ulex europaeus lectin and double stained with DIL-labeled ac-LDL and FITC-conjugated Ulex europaeus lectin (overlay). von Willebrand factor (vWF), VEGFR2, CD31 and double stained with VEGFR2 and CD31 (overlay) analysis were assessed by immunofluorescence (× 20).
Figure 2
Figure 2
Id1-RNAi-LV transfection silenced the mRNA and protein expression of Id1 and integrin α4 in EPCs. (A) Id1 and integrin α4 mRNA expression by real-time RT-PCR. Data are expressed as means ± S.E. **p < 0.01 vs control, #p < 0.05 vs ovarian cancer. (B) Typical Western blot images showing protein expression of Id1 and integrin α4 (β-actin is shown as a housekeeping control). (C) The graph showing the relative Id1 and integrin α4 protein levels normalized to β-actin. The results were expressed as the mean ± S.E.. **p < 0.01 vs. control, #p < 0.05 vs. ovarian cancer.
Figure 3
Figure 3
Effects of Id1-RNAi-LV and LY294002 on EPCs proliferation, migration, and adhesion functions. (A) Typical images of migration and adhesion as measured by transwell chamber and ECMatrix gel assay, respectively. (B) Effects of Id1-RNAi-LV and LY294002 on EPCs proliferation. **p<0.01 vs. control, #,$p<0.05 vs. ovarian cancer. (C-D) Accumulated data showing EPCs migration and adhesion functions. *p<0.05, **p<0.01 vs. control, #,$p<0.05 vs. ovarian cancer.
Figure 4
Figure 4
Effects of PI3K/Akt on the protein expression of Id1, integrin α4, p-Akt in EPCs. (A) Typical Western blot images showing protein expression of Id1, integrin α4, p-Akt and t-Akt (β-actin is shown as a housekeeping control). (B) The graph showing the relative Id1, integrin α4, and p-Akt protein levels normalized to actin. The results are expressed as the mean ± S.E. *p < 0.05 vs. control, #,$p < 0.05 vs. ovarian cancer.
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